Introduction

Gluconeogenesis is the biosynthesis of new glucose, (i.e. not glucose from glycogen).
This process is frequently referred to as endogenous glucose production (EGP). The production of glucose
from other carbon skeletons is necessary since the testes, erythrocytes and kidney medulla
exclusively utilize glucose for ATP production. The brain also utilizes large amounts of
the daily glucose consumed or produced via gluconeogenesis. However, in addition to glucose,
the brain can derive energy from ketone bodies
which are converted to acetyl-CoA and shunted into the
TCA cycle.
The primary carbon skeletons used for gluconeogenesis are derived from pyruvate,
lactate, glycerol, and the amino acids alanine and glutamine. The liver is the
major site of gluconeogenesis, however, as discussed below, the kidney and the
small intestine also have important roles to play in this pathway.

Synthesis of glucose from three and four carbon precursors is essentially a reversal of
glycolysis.
The relevant features of the pathway of gluconeogenesis
are diagrammed below:

Reactions of Gluconeogenesis: Gluconeogenesis from
two moles of pyruvate to two moles of 1,3-bisphosphoglycerate
consumes six moles of ATP. This makes the process of gluconeogenesis very costly
from an energy standpoint considering that glucose oxidation to two moles of
pyruvate yields two moles of ATP. The major hepatic substrates for gluconeogenesis
(glycerol, lactate, alanine, and pyruvate) are enclosed in red boxes for highlighting.
The reactions that take place in the mitochondria are pyruvate to OAA and OAA to malate.
Pyruvate from the cytosol is transported across the inner mitochondrial membrane by the
pyruvate transporter. Transport of pyruvate across the plasma membrane is catalyzed by the
SLC16A1 protein (also called the monocarboxylic acid transporter 1, MCT1) and transport across the outer mitochondrial membrane involves a voltage-dependent porin transporter. Transport across the inner mitochondrial membrane requires a heterotetrameric transport complex (mitochondrial pyruvate carrier) consisting of the MPC1 gene and MPC2 gene encoded proteins. Following
reduction of OAA to malate the malate is transported to the cytosol by the malate
transporter (SLC25A11). In the cytosol the malate is oxidized to OAA and the OOA then feeds
into the gluconeogenic pathway via conversion to PEP via PEPCK. The PEPCK reaction
is another site for consumption of an ATP equivalent (GTP is utilized in the PEPCK reaction). The reversal of the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reaction
requires a supply of NADH. When lactate is the gluconeogenic substrate
the NADH is supplied by the lactate dehydrogenase (LDH) reaction (indicated by the dashes lines), and it is
supplied by the malate dehydrogenase reaction when pyruvate and alanine are the substrates.
Secondly, one mole of glyceraldehyde-3-phosphate must be isomerized to DHAP
and then a mole of DHAP can be condensed to a mole of glyceraldehyde-3-phosphate
to form 1 mole of fructose-1,6-bisphosphate in a reversal of the aldolase reaction.
In hepatocytes the glucose-6-phosphatase (G6Pase) reaction allows the liver to
supply the blood with free glucose. Remember that due to the high Km
of liver glucokinase most of the glucose will
not be phosphorylated and will flow down its concentration gradient out of
hepatocytes and into the blood. ALT: alanine transaminase. PGAM1: phosphoglycerate mutase 1.
PGK1: phosphoglycerate kinase 1. TMI: triose isomerase. PGI: glucose-6-phosphate isomerase.
GPD1: cytosolic glycerol-3-phosphate dehydrogenase. F1,6BPase: fructose-1,6-bisphosphatase.

The three reactions of glycolysis that proceed with a large negative free
energy change are bypassed during gluconeogenesis by using different enzymes.
These three are the pyruvate kinase, phosphofructokinase-1 (PFK-1) and
hexokinase/glucokinase catalyzed reactions. In the liver, intestine, or kidney cortex,
the glucose-6-phosphate (G6P) produced by gluconeogenesis
can be incorporated into glycogen. In this case the third bypass occurs at
the glycogen phosphorylase catalyzed reaction. Since skeletal muscle lacks
glucose-6-phosphatase it cannot deliver free glucose to the blood and undergoes
gluconeogenesis exclusively as a mechanism to generate glucose for storage as glycogen.

Pyruvate to Phosphoenolpyruvate (PEP), Bypass 1

Conversion of pyruvate to PEP requires the action of two enzymes: pyruvate
carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK).

Pyruvate Carboxylase Reaction

The first reaction of bypass 1 utilizes the ATP and biotin-requiring enzyme pyruvate carboxylase, (PC).
PC is referred to as an ABC enzyme due to the role of ATP,
Biotin, and CO2 in its catalytic activities.
The CO2 utilized in the PC reaction is in the form of bicarbonate
(HCO3-) . As the name of the enzyme implies, pyruvate is carboxylated to form oxaloacetate
(OAA). PC is a somewhat unique enzyme in that it is one of only two metabolically
important enzymes that requires an obligate activator. In the
absence of its obligate activator, acetyl-CoA, PC is completely inactive. The
primary source of the acetyl-CoA required by PC comes from the oxidation of
fatty acids which are being delivered to the liver after release from adipose
tissue in response to fasting or stress. Another critical enzyme that functions
only in the presence of an obligate activator is carbamoylphosphate synthetase I (CPS I)
of the urea cycle.

Although the major function of PC is to drive precursor carbon atoms (from
pyruvate, lactate, and alanine) into the generation of endogenous glucose, the
production of oxaloacetate is also an important anaplerotic reaction since
it can be used to fill-up the TCA cycle.
Indeed, within the brain the primary function of PC is to ensure that glial
cells have sufficient oxaloacetate to drive the TCA cycle. In these cells, in
addition to energy generation, the TCA cycle is vital to the continued
generation of 2-oxoglutarate (α-ketoglutarate) which can be siphoned off the TCA cycle and
utilized for the synthesis of glutamate as a critical excitatory
neurotransmitter.

Like the other biotin-dependent carboxylating enzymes in mammals, PC is multi-functional and contains
three distinct enzymatic domains: the biotin carboxylase (BC) domain, the carboxyltransferase
(CT) domain, and the biotin carboxyl carrier protein (BCCP) domain. PC is
composed of four identical subunits generating an α4 homotetrameric enzyme.
The human PC gene is located on chromosome 11q13.2 and contains 19 exons
spanning 16 kbp of DNA. The expressed 4.2 kb PC mRNA encodes a protein of 1178
amino acids with a molecular weight of 129.6 kDa.

The reaction catalyzed by PC occurs in a two-step process. The first partial
reaction involves the fixation of CO2 to biotin that involves the BC and BCCP
domains. During this initial stage of the reaction, biotin is moved to interact with
the BC domain forming carboxybiotin. The carboxybiotin is brought into contact
with the carboxyltransferase domain resulting in the formation of carboxylated
biotin. This biotin carboxylase
reaction involves a carboxyphosphate intermediate formed directly from ATP
and bicarbonate. During the second step of the overall PC reaction,
carboxybiotin is decarboxylated and pyruvate is concurrently carboxylated
forming oxaloacetate.

Phosphoenolpyruvate Carboxykinase Reaction

The second enzyme in the conversion of pyruvate to PEP is PEP carboxykinase (PEPCK).
PEPCK requires GTP in the decarboxylation of OAA to yield PEP.
Since PC incorporated CO2 into pyruvate and it is subsequently
released in the PEPCK reaction, no net fixation of carbon occurs.
Human cells contain almost equal amounts of mitochondrial and cytosolic
PEPCK (designated PEPCK-m and PEPCK-c, respectively) so this second reaction can occur in either cellular compartment.
The PEPCK-c gene (official gene symbol: PCK1) is located on chromosome 20q13.31
and is composed of 10 exons encoding a protein of 622 amino acids. The PEPCK-m gene (official gene
symbol: PCK2) is located on chromosome 14q11.2 and is composed of 10 exons
encoding a 640 amino acid protein. The PCK2 gene is primarily expressed in the
liver, kidney, and intestine as would be expected for a major gluconeogenic
enzyme. The liver expresses the PCK1 and PCK2 genes at essentially equivalent
levels.

Transcription of the PCK1 and PCK2 genes has been shown to be regulated by insulin, glucagon,
and glucocorticoids. Regulation of PEPCK gene expression by glucagon is exerted
via the activation of the transcription factor, CREB (cAMP response
element-binding protein). When glucagon binds its receptor the result is
activation of adenylate cyclase with resultant increases in cAMP production. The
increased cAMP in turn activates PKA which, among numerous substrates,
phosphorylates CREB. Phosphorylated CREB then migrates to the nucleus where it
binds to a cAMP-response element (CRE) in the PEPCK genes activating their rate
of transcription. The stress hormone, cortisol, exerts a very similar effect on
PEPCK gene expression via binding of the cortisol-activated glucocorticoid
receptor to a glucocorticoid-response element (GRE) in the PEPCK genes.

For gluconeogenesis to proceed, the OAA produced by PC needs to be
transported to the cytosol. However, no transport mechanism exist for its'
direct transfer and OAA will not freely diffuse. Mitochondrial OAA can become cytosolic via three pathways:
(1) conversion to PEP as indicated above
through the action of the mitochondrial PEPCK; (2) transamination to aspartate; or
(3) reduction to malate, all of which are transported to the cytosol. Transport of mitochondrial PEP to the cytosol is carried out by the tricarboxylate transporter encoded by the SLC25A1 gene. The transport of malate to the cytosol is carried out by the transporter encoded by the
SLC25A11 gene. The transport of aspartate to the cytosol is carried out by either of two transporters, one is encoded by the
SLC25A12 gene and the other is encoded by the
SLC25A13 gene. In the context of the transamination of OAA to aspartate and the reduction of OAA to malate, there is a need for adequate levels of the other intermediates of the malate-aspartate shuttle to ensure these latter two reactions can continue.

The malate-aspartate shuttle. This shuttle is the principal mechanism for the movement of reducing equivalents
(in the form of NADH; highlighted in the red boxes) from the cytoplasm to the mitochondria. The glycolytic
pathway is a primary source of NADH. Within the mitochondria the electrons of
NADH can be coupled to ATP production during the process of oxidative phosphorylation. The electrons are
"carried" into the mitochondria in the form of malate. Cytoplasmic malate
dehydrogenase (MDH) reduces oxaloacetate (OAA) to malate while
oxidizing NADH to NAD+. Malate then enters the mitochondria where
the reverse reaction is carried out by mitochondrial MDH. Movement of
mitochondrial OAA to the cytoplasm to maintain this cycle requires it be
transaminated to aspartate (Asp, D) with the amino group being donated by
glutamate (Glu, E). The Asp then leaves the mitochondria and enters the
cytoplasm. The deamination of glutamate generates 2-oxoglutarate, 2-OG, (α-ketoglutarate)
which leaves the mitochondria for the cytoplasm. All the participants in the
cycle are present in the proper cellular compartment for the shuttle to
function due to concentration dependent movement. When the energy level of
the cell rises the rate of mitochondrial oxidation of NADH to NAD+
declines and therefore, the shuttle slows. GAPDH is glyceraldehyde-3-phosphate dehydrogenase.
AST is aspartate transaminase. SLC25A11 is the malate transporter and SLC25A13 is one of the
two mitochondrial aspartate/glutamate transporters.

If OAA is converted to PEP by mitochondrial PEPCK, it is transported to the
cytosol where it is a direct substrate for gluconeogenesis and nothing
further is required. Transamination of OAA to aspartate allows the aspartate to be
transported to the cytosol where the reverse transamination occurs
yielding cytosolic OAA. This transamination reaction requires continuous
transport of glutamate into, and 2-oxoglutatrate (α-ketoglutarate) out of, the mitochondrion.
Therefore, this process is limited by the availability of these other substrates.
Either of these latter two reactions will predominate when the substrate
for gluconeogenesis is lactate. Whether mitochondrial decarboxylation or
transamination occurs is a function of the availability of PEPCK or transamination intermediates.

Mitochondrial OAA can also be reduced to malate in a reversal of the
TCA cycle reaction catalyzed by malate dehydrogenase (MDH).
The reduction of OAA to malate requires NADH, which will be accumulating in
the mitochondrion as the energy charge increases.
The increased energy charge will allow cells to carry out the ATP costly
process of gluconeogenesis. The resultant malate is
transported to the cytosol where it is oxidized to OAA by cytosolic MDH which
requires NAD+ and yields NADH. The NADH produced during
the cytosolic oxidation of malate to OAA is utilized during the
glyceraldehyde-3-phosphate dehydrogenase reaction of gluconeogenesis.
The coupling of these two oxidation-reduction reactions is required to
keep gluconeogenesis functional when pyruvate is the principal
source of carbon atoms. The conversion of OAA to malate predominates
when pyruvate (derived from glycolysis or amino acid catabolism) is the
source of carbon atoms for gluconeogenesis. When in the cytoplasm, OAA is
converted to PEP by the cytosolic version of PEPCK.
Hormonal signals control the level of PEPCK protein as a means to regulate
the flux through gluconeogenesis (see below).

The net result of the PC and PEPCK reactions is:

Pyruvate + ATP + GTP + H2O ——> PEP + ADP + GDP + Pi + 2H+

Fructose-1,6-bisphosphate to Fructose-6-phosphate, Bypass 2

Fructose-1,6-bisphosphate (F1,6BP) conversion to fructose-6-phosphate (F6P) is the reverse of the rate limiting step of glycolysis.
The reaction, a simple hydrolysis, is catalyzed by fructose-1,6-bisphosphatase (F1,6BPase).
The existence of two distinct forms of F1,6BPase was recognized by comparison of
the kinetic and regulatory properties of the purified liver and muscle enzymes.
In addition, in patients with an
inborn error in the gene encoding the liver
F1,6BPase isoform, there is no reduction in skeletal muscle F1,6BPase activity.
This led to the characterization of two F1,6BPase genes in the human genome. One
expresses a liver version of the enzyme (gene symbol: FBP1) and the other a
muscle version of the enzyme (gene symbol: FBP2). The FBP1 gene is located on
chromosome 9q22.3 and is composed of 8 exons that encode a protein of 338 amino
acids. The FBP2 gene is located at the same chromosomal location as the FBP1
gene but is composed of 7 exons that encode a protein of 339 amino acids. The
liver and muscle F1,6BPase enzymes share 77% amino acid sequence identity.

Like the
regulation of glycolysis
occurring at the PFK-1 reaction, the F1,6BPase reaction is a major point of control of gluconeogenesis (see below).

Glucose-6-phosphate (G6P) to Glucose (or Glycogen), Bypass 3

Glucose-6-phosphate is converted to glucose through the action of enzymes of the glucose-6-phosphatase (G6Pase) family. The G6Pase reaction is also a simple
hydrolysis reaction like that of F1,6BPase.
Since the brain and skeletal muscle, as well as most non-hepatic tissues, lack G6Pase activity, any gluconeogenesis
that might occur in these tissues is not utilized
for blood glucose supply. In the kidney, muscle and especially the liver, G6P be shunted toward glycogen if blood
glucose levels are adequate.
The reactions necessary for glycogen synthesis are an alternate
bypass series of reactions.

The glucose-6-phosphatase activitites are membrane-associated multi-subunit complexes associated with the membranes of the endoplasmic reticulum, ER. The complexes are composed of a catalytic subunit and transporter proteins for the transport of glucose-6-phosphate, inorganic phosphate, and glucose across the membranes of the ER. The catalytic activity of G6Pases resides in a domain of the enzyme that is within the lumen of the ER, thus glucose-6-phosphate must first be transported into the ER for the phosphate to be removed. Humans express three distinct genes of the glucose-6-phosphatase family identified as G6PC, G6PC2, and G6PC3. The G6PC gene encodes the predominantly expressed functional phosphatase form of the glucose-6-phosphatase. The G6PC gene is located on chromosome 17q21.31 and is composed of 5 exons that encode a 357 amino acid protein. Only three human tissues express the G6PC gene, liver, kidney, and small intestine. Likewise, these are the only tissues that can
contribute to endogenous glucose production. Defects in the G6PC gene are associated with the glycogen
storage disease known as von Gierke disease (glycogen storage disease type Ia).

The ER membrane-localized glucose-6-phosphate transporter is encoded by the SLC37A4 gene with the encoded protein being identified as G6PT1. The ER membrane-localized phosphate transporter is encoded by the SLC17A3 gene and the encoded protein is identified as NPT4 (Na+-phosphate transporter 4). The SLC17A3 gene generates two alternatively spliced mRNAs with one mRNA encoding a 498 amino acid transporter that is localized to the apical membrane of epithelial cells of the proximal tubule of the kidney. The other SLC17A3 derived mRNA encodes a 420 amino acid transporter that is localized to the ER membrane. The transport of free glucose, from the lumen of the ER to the cytosol, most likely occurs through the actions of plasma membrane localized GLUT transporters (most likely GLUT2 in the liver) as they are transiting the ER on their way to the plasma membrane.

The G6PC2 gene is expressed in pancreatic islets but the encoded protein does not possess glucose-6-phosphatase activity. The G6PC2 gene is located on chromosome 2q31.1 and is composed of 5 exons that generate two alternatively spliced mRNA that encode different isoforms. The G6PC3 gene is located on chromosome 17q21.31 and is composed of 8 exons that generate two alternatively spliced mRNAs that encode distinct protein isoforms. The G6PC3 gene encoded protein is not involved in endogenous free glucose production but is believed to have a function in neutrophil activities. Although the G6PC3 encoded protein can hydrolyze phosphate from glucose-6-phosphate in vitro, the enzyme has a preference for other substrates in vivo.

Phosphorolysis of glycogen is carried out by glycogen phosphorylase,
whereas, glycogen synthesis
is catalyzed by glycogen synthase. The G6P produced from gluconeogenesis
can be used as a substrate for the synthesis of glycogen. In this case the
G6P is converted to glucose-1-phosphate (G1P) by phosphoglucomutase (PGM).
G1P is then converted to UDP-glucose (the substrate for glycogen synthase)
by UDP-glucose pyrophosphorylase, a reaction requiring hydrolysis of UTP.

Substrates for Gluconeogenesis

Lactate:

Lactate is a predominate source of carbon atoms for glucose
synthesis by gluconeogenesis. During anaerobic glycolysis in skeletal muscle,
pyruvate is reduced to lactate by lactate dehydrogenase (LDH).
This reaction serves two critical functions during anaerobic glycolysis.
First, in the direction of lactate formation the LDH reaction requires NADH
and yields NAD+ which is then available for use by the
glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis.
These two reaction are, therefore, intimately coupled during anaerobic glycolysis.
Secondly, the lactate produced by the LDH reaction is released to the blood
stream and transported to the liver where it is converted to glucose.
The glucose is then returned to the blood for use by muscle as an energy
source and to replenish glycogen stores. This cycle is termed the
Cori cycle.

The Cori Cycle: This cycle involves the utilization of lactate,
produced by glycolysis in non-hepatic tissues, (such as muscle and erythrocytes) as a
carbon source for hepatic gluconeogenesis. In this way the liver can
convert the anaerobic byproduct of glycolysis, lactate, back into more glucose
for reuse by non-hepatic tissues. Note that the gluconeogenic leg of
the cycle (on its own) is a net consumer of energy, costing the body 4 moles of
ATP more than are produced during glycolysis. Therefore, the cycle cannot be sustained indefinitely.

Pyruvate:

Pyruvate, generated in muscle and other peripheral tissues, can be
transaminated to alanine which is returned to the liver for gluconeogenesis.
The transamination reaction requires an α-amino acid as donor of
the amino group, generating an α-keto acid in the process.
This pathway is termed the glucose-alanine cycle.
Although the majority of amino acids are degraded in the liver some are deaminated in muscle.
The glucose-alanine cycle is, therefore, an indirect mechanism for
muscle to eliminate nitrogen while replenishing its energy supply.
However, the major function of the glucose-alanine cycle is to allow
non-hepatic tissues to deliver the amino portion of catabolized amino acids
to the liver for excretion as urea. Within the liver the alanine is converted back to pyruvate and used as a gluconeogenic substrate
(if that is the hepatic requirement) or oxidized in the TCA cycle.
The amino nitrogen is converted to urea in the
urea cycle and excreted by the kidneys.

The glucose-alanine cycle: This cycle is used primarily as a
mechanism for skeletal muscle to eliminate nitrogen while replenishing its energy supply.
Glucose oxidation produces pyruvate which can undergo transamination
to alanine. This reaction is catalyzed by alanine transaminase,
ALT (ALT used to be referred to a serum glutamate-pyruvate transaminase, SGPT).
Additionally, during periods of fasting, skeletal muscle
protein is degraded for the energy value of the amino acid carbons and
alanine is a major amino acid in protein. The alanine then enters the
blood stream and is transported to the liver. Within the liver alanine
is converted back to pyruvate which is then a source of carbon atoms for gluconeogenesis.
The newly formed glucose can then enter the blood for delivery back to
the muscle. The amino group transported from the muscle to the liver
in the form of alanine is converted to urea in the urea cycle and excreted.

Amino Acids:

All of the amino acids present in proteins, excepting leucine and lysine, can be degraded to TCA cycle intermediates as discussed in the
metabolism of amino acids.
This allows the carbon skeletons of the amino acids to be
converted to those in oxaloacetate and subsequently into pyruvate.
The pyruvate thus formed can be utilized by the gluconeogenic pathway.
When glycogen stores are depleted, in muscle during exertion and
liver during fasting, catabolism of muscle proteins to amino acids contributes
the major source of carbon for maintenance of blood glucose levels. Of all the
amino acids utilized for gluconeogenesis, glutamine is the most important as
this amino acid is critical for glucose production by the kidneys and small
intestine.

Glutamine

Glutamine is the sole source of carbon atom for the gluconeogenesis pathway carried
out in the kidney and the small intestine. In these two tissues, glutamine is first deaminated to glutamate via the action of glutaminase. The glutamate is then further deaminated,
via the action of the enzyme glutamate dehydrogenase, yielding 2-oxoglutarate (α-ketoglutarate). The
2-oxoglutarate can then enter the TCA cycle where it is eventually converted to malate. As described earlier, malate can be transported out of the mitochondria and oxidized to
oxaloacetate via the action of cytoplasmic malate dehydrogenase. The oxaloacetate is then
converted to PEP via the action of the cytoplasmic version of phosphoenolpyruvate
carboxykinase (PEPCK-c). Alternatively, malate can be oxidized to oxaloacetate within the mitochondria
then the action of mitochondrial PEPCK (PEPCK-m) can convert the oxaloacetate to PEP.
If this pathway is utilized the PEP is
transported to the cytosol for gluconeogenesis.

Glycerol:

Oxidation of fatty acids yields enormous amounts of energy on a molar basis,
however, the carbons of the fatty acids cannot be utilized
for net synthesis of glucose. The two carbon unit of acetyl-CoA derived from
β-oxidation
of fatty acids can be incorporated into the TCA cycle, however,
during the TCA cycle two carbons are lost as CO2.
Thus, explaining why fatty acids do not undergo net conversion to carbohydrate. However, the glycerol backbone that is released from adipocytes following hormone-induced triglyceride breakdown can be used for gluconeogenesis.
This requires phosphorylation of the glycerol to glycerol-3-phosphate by glycerol
kinase within hepatocytes. Following formation of glycerol-3-phosphate it is oxidized to dihydroxyacetone phosphate (DHAP)
by cytosolic glycerol-3-phosphate dehydrogenase 1 (GPD1). The glycerol backbone of adipose tissue stored triacylglycerides is
ensured of being used as a gluconeogenic substrate by the liver since adipocytes lack glycerol kinase. In fact adipocytes require a basal level
of glycolysis in order to provide them with DHAP as an intermediate
in the synthesis of triacylglycerides. The GPD1 reaction
is the same as that used in the transport of cytosolic reducing
equivalents into the mitochondrion for use in oxidative phosphorylation.
This transport pathway is called the glycerol-phosphate shuttle.

The glycerol phosphate
shuttle. This shuttle is a secondary mechanism for the transport of electrons from
cytosolic NADH to mitochondrial carriers of the oxidative phosphorylation
pathway. The primary cytoplasmic NADH electron shuttle is the malate-aspartate shuttle. Two
enzymes are involved in this shuttle. One is the cytosolic version of GPD (GPD1) which utilizes NADH as a co-enzyme. The second is the mitochondrial form of
the enzyme (GPD2) which utilizes FAD+ as co-enzyme. The net
result is that there is a continual conversion of the glycolytic
intermediate, DHAP and glycerol-3-phosphate with the concomitant transfer of
the electrons from reduced cytosolic NADH to mitochondrial oxidized FAD+.
Since the electrons from mitochondrial FADH2 feed into the
oxidative phosphorylation pathway
at coenzyme Q (as opposed to NADH-ubiquinone oxidoreductase [complex
I]) only 2 moles of ATP will be generated from glycolysis. GAPDH is glyceraldehyde-3-phoshate dehydrogenase.

Propionate:

Oxidation of fatty acids with an odd number of carbon atoms
and the oxidation of some amino acids
generates as the terminal oxidation product, propionyl-CoA.
Propionyl-CoA is converted to the TCA intermediate, succinyl-CoA.
This conversion is carried out by the ATP-requiring enzyme,
propionyl-CoA carboxylase then methylmalonyl-CoA epimerase and finally the
vitamin B12
requiring enzyme, methylmalonyl-CoA mutase. The utilization of propionate
in gluconeogenesis only has quantitative significance in ruminants.

Conversion of Propionyl-CoA to Succinyl-CoA

Propionyl-CoA carboxylase functions as a heterododecameric enzyme (subunit composition: α6β6) and the two different subunits are encoded by the PCCA and PCCB genes respectively. The PCCA gene is located on chromosome 13q32 and is composed of 27 exons that generates three alternatively spliced mRNAs. The PCCB gene is located on 3q21–q22 and is composed of 17 exons that generate two alternatively spliced mRNAs. Methylmalonyl-CoA epimerase is encoded by the MCEE gene located on chromosome 2p13.3 and is composed of 4 exons that encode a 176 amino acid protein. Methylmalonyl-CoA mutase is encoded by the MUT gene located on chromosome 6p12.3 and is composed of 13 exons that encode a protein of 750 amino acids. Mutations in the MUT gene are one cause of the methylmalonic acidemias. Mutations in either the PCCA or PCCB gene are associated with propionic acidemia associated with severe ketoacidosis. The original identification of a child suffering from propionyl-CoA deficiency was in 1961. This child suffered frequent episodes of severe ketoacidosis, all of which were precipitated by protein ingestion. Blood and urine analysis demonstrated marked elevations in glycine levels. These initial laboratory studies lead to the disorder being called ketotic hyperglycinemia.
However, there is no defect in glycine metabolism with inherited mutations in PCCA or PCCB. The clinical hallmark of the disease is severe ketoacidosis of an episodic nature.

Role of Intestinal Gluconeogenesis and Control of Feeding Behaviors

Intestinal Gluconeogenesis

The gut, in particular the small intestine, plays a critical role in the
uptake and delivery of glucose from the diet. As such, the gut plays a central
role in the overall regulation of glucose homeostasis. Glucose uptake from the
lumen of the gut and trans-epithelial transport to the portal circulation had
been shown to occur via action of two distinct glucose transporters. First,
glucose is taken up from the intestinal lumen through the action of the
sodium-dependent glucose transporter-1 (SGLT-1) then it is transported into the
portal blood via the action of the facilitated glucose transporter GLUT2 present
in the basolateral membrane. Evidence has also indicated that GLUT2 present in
the apical (luminal) membrane of enterocytes was involved in glucose uptake.
However, GLUT2 is not present in the apical membrane in the absence of a glucose
load. The mechanism of GLUT2 presentation in the apical membrane involves a
glucose-induced translocation of GLUT2 to this membrane. Thus, glucose uptake by
the small intestine enhances additional uptake by promoting presentation of an
additional transporter in the apical membrane.

The small intestine also utilizes glucose, obtained from the diet or from the
blood, for energy production. Recently it was shown that the intestine is able
to utilize glutamine for energy with the same efficiency as glucose. Indeed,
glutamine has been considered to be a major energy substrate for this organ. Of
additional, significance, and only recently having been determined, is the role
of intestinal gluconeogenesis in overall endogenous glucose production (EGP). A
little over 10 years ago, molecular analysis allowed for the characterization of
the expression of glucose-6-phosphatase (G6Pase) within enterocytes of the small
intestine. Expression of G6Pase thus, confers upon the intestine, the ability to
carry out gluconeogenesis. Now it is known that glutamine serves as the major precursor of glucose
formed within the small intestine. The genes for both G6Pase and the cytosolic
form of phosphoenolpyruvate carboxykinase (PEPCK-c)
are controlled by insulin in the small intestine similarly to the regulation of
these genes in the liver. The presence of G6Pase within the small intestine also
plays a role in the export of glucose to the portal circulation. This can either
be dietary glucose, glucose released from intestinal glycogen stores, or glucose
produced via gluconeogenesis. This glucose export mechanism is dependent on the
previous phosphorylation of glucose by hexokinases followed by G6Pase-mediated dephosphorylation.

Pathways of gluconeogenesis in the small intestine and coupling to gluconeogenic substrate delivery to the liver. Glucose and glutamine arrive in intestinal enterocytes either from the diet or the arterial blood
supply as depicted. The carbon atoms of glutamine serve as the major substrate for
intestinal gluconeogenesis via the two-step process catalyzed by glutaminase and
alanine transaminase (ALT). The resultant 2-oxoglutarate (α-ketoglutarate)
is converted to oxaloacetate (OAA) and then to phosphoenolpyruvate (PEP) which is then
diverted into the gluconeogenic pathway. Glucose that enters the enterocyte can be oxidized
to pyruvate via glycolysis and then the carbons of pyruvate can be reduced to lactate or
transaminated to alanine, both of which can serve as major gluconeogenic substrate
in the liver following delivery via the portal circulation. The contributions of intestinal
glycerol and glucose from glycogen to the role of the intestine ion overall glucose homeostasis
is also depicted. GPD is glycerol-3-phosphate dehydrogenase. PGM is phosphoglycerate mutase.
G6P is glucose-6-phosphate. G3P is glyceraldehyde-3-phosphate. LDH is lactate dehydrogenase.

The importance of intestinal gluconeogenesis, to overall EGP, has been
demonstrated both in experimental animals (mice with specific knockout of
PEPCK-c in the liver) and in humans in the anhepatic phase during liver
transplantation. In mice without hepatic PEPCK-c there is an efficient
adaptation to fasting conditions such that blood glucose levels decrease by only 30%.
In addition, in these mice, and humans undergoing liver transplant, there occurs
a significant increase in plasma glutamine concentration. These observations stressed the likely role of
the kidney and/or intestine in glucose production, because
glutamine is a major glucose precursor in the kidney and
the small intestine, but not in the liver. The role of the intestine in this
glucose control was demonstrated by the fact that in these experimental
conditions there is no observable difference in glucose concentration between
arterial and portal blood.

During periods of fasting the small intestine accounts for approximately 20%
of EGP by 48hrs and up to 35% by 72hrs. However, expression of the key gluconeogenic
genes, G6Pase and PEPCK-c, is dependent on plasma insulin concentrations, and these do not change
throughout these time frames of fasting. Yet expression of both of these genes
is seen to increase within intestinal cells between 24 hrs and 48 hrs of the
initiation of fasting. The promoter region of the G6Pase gene (G6PC), containing the presumed TATA box (TATAAAA,
located -31 to -25 bp upstream of the transcription start site), also constitutes a putative binding
site for transcription factors of the caudal-related homeobox (CDX) family. In adult mammals, the
CDX genes are exclusively expressed in the gut, where they are involved
in the differentiation of both the crypt-villus and anteroposterior
axis. The TATAAAA element in the G6PC gene is indeed responsible for its transactivation by CDX1
(but not CDX2) since disruption of the sequence strongly blunts basal transcription and CDX1 transactivation of the gene.

Intestinal Gluconeogenesis and Feeding Behaviors

Protein-rich diets are known to reduce hunger and
subsequent food intake in both humans and experimental animals. In addition, protein-rich, carbohydrate-free diets have been shown to
strongly induce the expression of G6Pase, PEPCK-c, and glutaminase in the intestine.
In addition, the gut releases glucose to the portal circulation following the
intake of a protein-rich, carbohydrate-free diet. The rate of glucose release by the gut can be
estimated to provide about 15% to 20% of EGP in protein-fed
experimental animals. Significantly, this level of glucose release from the gut is sufficient to account for the
level of reduction in food intake observed in protein-fed animals, where an equivalent infusion of
glucose into the portal vein of the control animals also decreased food intake and by a comparable value.
Although this glucose, derived by intestinal gluconeogenesis, does not increase
overall EGP this is because the liver adapts by decreasing
its own level of gluconeogenesis while also increasing glycogen storage.

That intestinal gluconeogenesis is indeed crucial in the control
of food intake by dietary protein was established with
the use mice in which expression of the G6Pase gene was
specifically and conditionally abolished in the intestine. When these mice are
fed a protein-rich, carbohydrate-free diet they do not exhibit a decrease in their
level of food intake such as is seen in control mice on the same diet. The same
loss of satiety induction by protein-rich diets or portal glucose infusion is
seen in animals whose portal vein afferent nerve connections are chemically
or surgically destroyed. Afferent nerves send signals from body locations to the brain.
These types of studies demonstrate that portal sensing
of intestinal gluconeogenesis is a key mechanism in the
satiety effect induced by dietary protein.
Brain areas involved in the control of feeding behaviors include the brain stem
and the hypothalamus. For a detailed discussion of the role of the
hypothalamus in the control of feeding behaviors visit the Gut-Brain
Interactions page. In experimental animals fed protein-rich diets or who
have had glucose infusions into the portal vein, neuronal activation is observed
in several hypothalamic nuclei involved in feeding behavior regulation including
the arcuate nucleus (ARC), dorsomedial nucleus (DMN), ventromedial nucleus
(VMN), and paraventricular nucleus (PVN). In similar experiments in animals whose gut afferent circuits
have been destroyed, there is no increase in neuronal activity following portal
vein glucose infusion or consumption of protein-rich diets. In addition to the
effects, on feeding behavior, of intestinal glucose (via gluconeogenesis)
delivery to the portal circulation, numerous gut hormones are known to be involved in the control of hunger sensations
and do so, in part, via gastrointestinal afferent circuits.
The hunger-modulating effects initiated by the release of meal-dependent
gut hormones, including cholecystokinin (CKK), glucagon-like peptide-1 (GLP-1),
and PYY3-36, are all strongly attenuated by disrupting nerve
circuitry between the gastrointestinal and central nervous systems.

Role of Renal Gluconeogenesis

Although the liver has the critical role of maintaining blood glucose
homeostasis and therefore, is the major site of gluconeogenesis, the kidney
plays an important role. During periods of severe hypoglycemia that occur under
conditions of hepatic failure, the kidney can provide glucose to the blood via
renal gluconeogenesis. In the renal cortex, glutamine is the preferred substance
for gluconeogenesis.

Glutamine is produced in high amounts by skeletal muscle
during periods of fasting as a means to export the waste nitrogen resulting from
amino acid catabolism. Through the actions of transaminases, a mole of waste
ammonia is transferred to 2-oxoglutarate (α-ketoglutarate) via the glutamate dehydrogenase
catalyzed reaction yielding glutamate. Glutamate is then a substrate for
glutamine synthetase which incorporates another mole of waste ammonia generating
glutamine (see the Nitrogen Metabolism page
for more details). The glutamine is then transported to the kidneys where the
reverse reactions occur liberating the ammonia and producing 2-oxoglutarate which can enter the
TCA cycle
and the carbon atoms diverted to gluconeogenesis via oxaloacetate.
This process serves two important functions. The ammonia (NH3) that is liberated
spontaneously ionizes to ammonium ion (NH4+) and is excreted in the urine
effectively buffering the acids in the urine. In addition, the glucose that is
produced via gluconeogenesis can provide the brain with critically needed energy.

Regulation of Gluconeogenesis

Obviously the regulation of gluconeogenesis will be in direct contrast
to the regulation of glycolysis. In general, negative effectors of
glycolysis are positive effectors of gluconeogenesis. Regulation of the
activity of PFK-1 and F1,6BPase is the most significant site for
controlling the flux toward glucose oxidation or glucose synthesis.
As described in control of glycolysis,
this is predominantly controlled by fructose-2,6-bisphosphate, F2,6BP which
is a powerful negative allosteric effector of F1,6Bpase activity.

Regulation of glycolysis and gluconeogenesis by fructose 2,6-bisphosphate (F2,6BP).
The major sites for regulation of glycolysis and gluconeogenesis are the phosphofructokinase-1 (PFK-1) and
fructose-1,6-bisphosphatase (F-1,6-BPase) catalyzed reactions. PFK-2 is the kinase activity and F-2,6-BPase
is the phosphatase activity of the bi-functional regulatory enzyme, phosphofructokinase-2/fructose-2,6-bisphosphatase.
PKA is cAMP-dependent protein kinase which phosphorylates PFK-2/F-2,6-BPase turning on the phosphatase activity.
Green arrows indicate positive actions. Red T-lines represent inhibitory actions.

The level of F2,6BP will decline in hepatocytes in response to glucagon
stimulation as well as stimulation by catecholamines.
Each of these signals is elicited through activation of cAMP-dependent
protein kinase (PKA). One substrate for PKA is PFK-2, the bifunctional
enzyme responsible for the synthesis and hydrolysis of F2,6BP. When
PFK-2 is phosphorylated by PKA it acts as a phosphatase leading to the
dephosphorylation of F2,6BP with a concomitant increase in F1,6Bpase
activity and a decrease in PFK-1 activity. Secondarily, F1,6Bpase activity
is regulated by the ATP/ADP ratio. When this is high, gluconeogenesis can proceed maximally.

Gluconeogenesis is also controlled at the level of the pyruvate to PEP bypass.
The hepatic signals elicited by glucagon or epinephrine lead
to phosphorylation and inactivation of pyruvate kinase (PK) which will allow for an
increase in the flux through gluconeogenesis. PK is also
allosterically inhibited by ATP and alanine. The former signals adequate energy and
the latter that sufficient substrates for gluconeogenesis are available.
Conversely, a reduction in energy levels, as evidenced by increasing concentrations of
ADP, lead to inhibition of both PC and PEPCK. Activation
of PC occurs through interaction with acetyl-CoA. Indeed, PC is catalytically
inactive in the absence of acetyl-CoA. This fact defines the role of acetyl-CoA as an
obligate activator of PC. These regulations occur on a short time scale,
whereas long-term regulation can be effected at the level of PEPCK.
The amount of this enzyme increases in response to prolonged glucagon stimulation.
This situation would occur in a starving individual or someone with an inadequate diet.

Whereas glucagon actions results in increased levels of cAMP and subsequent
activation of gluconeogenesis, insulin action exerts the opposite effect. The
mechanisms by which insulin turns off gluconeogenesis are complex.
Reduction in the level of cAMP is exerted via the insulin-mediated activation
of phosphodiesterase (PDE3B) which hydrolyzes cAMP to AMP. At the level of the
regulation of genes involved in gluconeogenesis, cAMP signaling leads to
phosphorylation of the transcription factor CREB at Ser133. When phospho-CREB
binds to the cAMP response element (CRE) of a target gene it results in the recruitment
of the coactivators CBP and p300 (which are closely related). This complex activates gene
expression through their intrinsic histone acetyltransferase activity and through recruitment of other coactivator molecules.
The coactivator CBP is a target of insulin-dependent phosphorylation at Ser436.
This residue, which is adjacent to the CREB-binding domain (CREB-BD), is phosphorylated by a phosphoinositol-3-kinase
(PI3K)−dependent insulin signaling pathway, and is not conserved in the related cofactor p300.

The transcriptional coactivator PGC-1α
(peroxisome proliferator-activated receptor-γ coactivator-1α)
functions as a central regulator of gluconeogenesis by binding to several factors,
including the glucocorticoid receptor, hepatic nuclear factor 4α
(HNF4α) and a forkhead transcription factor (FOXO1) upstream of several genes encoding gluconeogenic enzymes.
One mechanism by which insulin signaling antagonizes gluconeogenesis is through phosphorylation of
FOXO1 and its subsequent exclusion from the nucleus. Expression of PGC-1α, however, is also regulated
by cAMP through a well-defined CRE present in the promoter region of the PGC-1α gene.